Streptococcus mutans, the etiologic agent responsible for dental caries, has been reported to contain antigens cross-reactive with human cardiac and skeletal muscle. These finds warrant caution in design and testing of an anti-caries vaccine and demand further investigations of the immunochemistry of the walls and membranes of this dental pathogen. The present project focuses on the cell membrane, site of localization of cross-reactive antigens (CRA) in beta-hemolytic streptococci and proposed site in this species. Membranes will be prepared by first converting cells to protoplasts using the muramidase, mutanolysin. Membranes from cells grown to mid-logarithmic and early-stationary phases will be analyzed for sugar, sugar alcohol, amino acid, amino sugar and fatty acid contents. Individual proteins will be detected by isoelectric focusing and SDS-PAGE. The biologic nature of component membrane proteins will be determined by crossed-immunoelectrophoresis (CIE): enzymes by zymogram analyses, glycoproteins by immunoaffinoelectrophoresis and specific antigens by intermediate gel techniques. CRA's in membranes (or enzyme digests of purified walls) will be identified by using anti-human heart and anti-S. pyogenes immunoglobulins in CIE. Cellular localizations of CRA's will be confirmed by adsorptions of the positive, heterologous antiserums with membranes and walls. Combination techniques (e.g. SDS-PAGE-CIE and cross-immunoisoelectric focusing) will allow determinations of molecular weights and isoelectric points of membrane proteins. The molecular architecture of membranes will be determined by adsorbing homologous antiserums with protoplasts and purified membranes to localize proteins as outer-surface components or inner (cytoplasmic)-surface moieties. Radioiodination of protoplasts will confirm the identity of outer surface proteins. CRA's will be isolated and purified by immunoaffinity column chromatography of crude membrane (wall) extracts. Products will be analyzed for chemical compositions, molecular weights and isoelectric points. Purified CRA's will be tested by indirect immunofluorescence and indirect radioimmunoassay for relative abilities to bind directly to heart tissue.